Viruses and their hosts have co-evolved an intricate interplay that is delicately balanced during the course of infection. The value of exploring such a natural relationship, rather than the mismatched experimental systems that currently prevail, is becoming increasingly clear. This program will focus squarely upon ectromelia virus (ECTV), a genuine mouse pathogen with several attractive properties. First, routes of transmission are similar to those of other orthopoxviridae in their natural hosts. Second, mousepox disease is remarkably similar to that of human monkeypox and smallpox. Third, some mouse strains are resistant to ECTV while others are highly susceptible, analogous to natural variations within human populations. None of this is true for vaccinia virus (VACV), an orthopoxvirus of unknown origin, frequently used in mouse models of immunity, and made all the more suspect by preliminary results presented below. Headed by three PIs with extensive experience in host defense against poxviruses, the program will comprehensively dissect the interplay between ECTV and resistant/susceptible strains of mice, referencing to VACV when appropriate. Project 1 will examine the cells and cytokines of the innate system that play critical roles in keeping the virus in check at the site of infection. Project 2 continues inspection of innate/natural immunity by examining the mechanisms by which type 1 interferons limit spread of the virus beyond the draining lymph node. Finally, Projects 2 and 3 will investigate all three major arms of the adaptive immune system to understand how they defend against primary infection and establish protection from subsequent challenge. All three projects will examine strategies by ECTV to thwart these different levels of host defense. Administrative, Biological Reagent, and Imaging Core Components will provide a wide range of support to the program, with the latter two carrying out their own exploratory projects. Our goal with this hypothesis-driven, highly interactive program is to develop an understanding of virus-host relationships that provides a framework for many other natural virus infections. PROJECT 1: THE INNATE IMMUNE RESPONSE TO MOUSEPOX AT THE SITE OF INFECTION (Norbury, C) PROJECT 1 DESCRIPTION (provided by applicant): During a natural virus infection small doses of infectious virus are deposited at a peripheral infection site and then a race ensues, in which the replicating virus attempts to outpace the host's immune system. In the early phases of infection, the innate immune system must contain the infection prior to the development of an adaptive response. In Project 1 we will examine the mechanisms that are used by the innate immune system to contain infection with mousepox, a lethal mouse disease caused by ectromelia virus (ECTV), an exclusive mouse pathogen. This system is unique because it allows us to examine the innate response in susceptible and resistant mouse strains. The three Specific Aims will examine the cells that are required to slow the systemic spread of ECTV at the site of infection, the chemoattractants that mediate their migration to the site of infection and the cell biological mechanisms that are used by both the virus and the immune system during virus-cell interaction. In Aim 1 we will characterize the cellular infiltrate to the site of ECTV infection in resistant or susceptible mice and identify the innate immune effector cell types that are required to slow the systemic spread of ECTV and allow the development of an adaptive response that can clear the infection. In this aim we will also examine the effector functions that are required by innate immune cells to retard ECTV infection. In Aim 2 we will determine the chemokines and chemokine receptors expressed at the site of ECTV infection in resistant or susceptible mice, and the chemokines that are essential to attract innate effector cells that slow replication and spread of the virus. We will also study the role of immune modifiers of cellular migration encoded by ECTV in the innate response to the virus, and will identify the targets of these genes in vivo. In Aim 3 we will study the interaction of ECTV and innate immune cells in vitro, focusing primarily upon macropinocytosis, which has recently been described as the mode of infection of orthopoxviruses. Macropinocytosis has an important role in the sampling of extracellular solute for initiation of an adaptive immune response and we will examine its contribution to sampling of the environment for initiation of an innate response. We will also examine the trafficking to macropinosomes of TLR9, an innate receptor that is required for survival from ECTV challenge. The results from this Project will provide a comprehensive picture of the innate response to a peripheral virus infection.